Comprehensive profiling of translation initiation in influenza virus infected cells
Comprehensive profiling of translation initiation in influenza virus infected cells
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DOI：
10.1371/journal.ppat.1007518
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发表时间：
2019-01-01
期刊：
影响因子：
6.7
通讯作者：
Subramaniam, Arvind R.
中科院分区：
文献类型：
--
作者：
Machkovech, Heather M.;Bloom, Jesse D.;Subramaniam, Arvind R.
翻译可以启动在交替的，非规范的启动密码子响应应激刺激在哺乳动物细胞。最近的研究表明，病毒感染和抗病毒反应改变了翻译起始位点，在某些情况下，导致新的免疫表位的产生。在这里，我们系统地研究了流感病毒感染细胞中交替翻译起始的程度和影响。我们进行了进化分析，表明在适应哺乳动物宿主的流感病毒谱系中，选择了CUG密码子的非规范起始。然后，我们使用起始抑制剂乳酸米多霉素的核糖体分析，实验地描绘了感染流感病毒的人肺上皮细胞系的翻译起始位点。我们在流感mrna中确定了几个交替起始的候选位点，所有这些位点都发生在AUG密码子上，这些密码子位于标准起始密码子的下游。这些候选下游起始位点之一从N1神经氨酸酶蛋白的n端截断14个氨基酸，导致其细胞质尾部和部分跨膜结构域的丢失。这种截断的神经氨酸酶蛋白在流感病毒感染期间在细胞表面表达，具有酶活性，并且在大多数N1病毒谱系中是保守的。在流感感染或抗病毒应答期间，我们没有在宿主转录本上检测到更高水平的交替翻译起始，但抗病毒应答诱导的宿主转录本亚群在交替起始位点上富集。总之，我们的结果系统地绘制了流感病毒感染期间翻译起始的景观，并阐明了形成这一景观的进化力量。当流感等病毒感染细胞时，宿主和病毒mrna都被翻译成蛋白质。在这里，我们研究了这些mrna中在流感感染期间启动蛋白质翻译的位点。特别是，我们探讨了一些翻译是否开始于密码子，而不是用于产生每个基因的初级蛋白产物的规范密码子。通过计算分析，我们发现哺乳动物流感病毒进化为减少可以启动这种替代翻译起始产物的密码子数量。接下来，我们使用核糖体分析的综合实验策略来确定所有流感和宿主mrna的翻译起始位点。我们在流感病毒和宿主mrna上发现了许多交替起始的位点。我们详细研究了流感mRNA上的一个这样的替代起始位点，并表明它编码一种病毒蛋白的功能性和以前未表征的变体。我们还发现有证据表明，宿主细胞在病毒感染期间表达的mrna在非规范起始密码子处富集用于翻译起始。总的来说，这些结果表明交替翻译起始在流感感染期间产生的病毒和宿主蛋白的形成中起作用。
Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactimidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. We do not detect globally higher levels of alternate translation initiation on host transcripts upon influenza infection or during the anti-viral response, but the subset of host transcripts induced by the anti-viral response are enriched for alternate initiation sites. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.Author summary When viruses such as influenza infect cells, both host and viral mRNAs are translated into proteins. Here we investigate the sites in these mRNAs that initiate protein translation during influenza infection. In particular, we explore whether some of this translation initiates at codons other than the canonical ones used to produce the primary protein product of each gene. Using computational analyses, we find that mammalian influenza viruses evolve to reduce the number of codons that can initiate such alternate translation initiation products. We next use the comprehensive experimental strategy of ribosome profiling to identify sites of translation initiation across all influenza and host mRNAs. We find a number of sites of alternate initiation on both influenza and host mRNAs. We study in detail one such alternate start site on an influenza mRNA, and show that it encodes a functional and previously uncharacterized variant of a viral protein. We also find evidence that the mRNAs that host cells express at higher levels during viral infection are enriched for translation initiation at non-canonical start codons. Overall, these results suggest that alternate translation initiation plays a role in shaping the repertoires of both viral and host proteins that are produced during influenza infection.